Mitochondrial Localization of 3β-Hydroxysteroid Dehydrogenase 5-ene Isomerase in Interrenals of the ToadBufo arenarumH

The enzymatic activity of 3β-hydroxysteroid dehydrogenase 5-ene isomerase (3βHSD/I) catalyzes an essential step in the biosynthesis of steroid hormones including progesterone, mineralocorticoids, glucocorticoids, estrogens, and androgens. Its subcellular localization in steroidogenic tissues is usually considered to be mainly microsomal. The present study demonstrates that in the interrenal ofBufo arenarumH., 3βHSD/I activity localizes in mitochondria and micromes. It also shows that the two distinct pathways to aldosterone previously demonstrated for interrenals ofB. arenarumH. exhibit differential subcellular localizations, microsomal for the 4-ene route and mitochondrial for the 5-ene route. Kinetic constants of 3βHSD/I were determined for the oxidation of pregnenolone and the recently described 3β-hydroxy analogue of aldosterone (3βAA). The preferred substrate of the mitochondrial 3βHSD/I enzyme was 3βAA (Km=0.7 μMand 14.0 μMfor 3βAA and pregnenolone, respectively). However, the microsomal enzyme has a greater affinity for pregnenolone (Km=0.8 μM) than for 3βAA (Km=17.0). Enzymes from both localizations have similar nucleotide (NAD+) requirements, activities being higher in summer. This dual localization opens novel possibilities for the regulation of interrenal functions.